Glioblastoma (GBM) is the most common primary malignant cancer of the central nervous system. Insufficient oxygenation (hypoxia) has been linked to GBM invasion and aggression, leading to poor ...patient outcomes. Hypoxia induces gene expression for cellular adaptations. However, GBM is characterized by high intertumoral (molecular subtypes) and intratumoral heterogeneity (cell states), and it is not well understood to what extent hypoxia triggers patient-specific gene responses and cellular diversity in GBM. Here, we surveyed eight patient-derived GBM stem cell lines for invasion phenotypes in 3D culture, which identified two GBM lines showing increased invasiveness in response to hypoxia. RNA-seq analysis of the two patient GBM lines revealed a set of shared hypoxia response genes concerning glucose metabolism, angiogenesis, and autophagy, but also a large set of patient-specific hypoxia-induced genes featuring cell migration and anti-inflammation, highlighting intertumoral diversity of hypoxia responses in GBM. We further applied the Shared GBM Hypoxia gene signature to single cell RNA-seq datasets of glioma patients, which showed that hypoxic cells displayed a shift towards mesenchymal-like (MES) and astrocyte-like (AC) states. Interestingly, in response to hypoxia, tumor cells in IDH-mutant gliomas displayed a strong shift to the AC state, whereas tumor cells in IDH-wildtype gliomas mainly shifted to the MES state. This distinct hypoxia response of IDH-mutant gliomas may contribute to its more favorable prognosis. Our transcriptomic studies provide a basis for future approaches to better understand the diversity of hypoxic niches in gliomas.
Glioblastoma (GBM), a highly lethal brain cancer, is notorious for immunosuppression, but the mechanisms remain unclear. Here, we documented a temporospatial patterning of tumor-associated myeloid ...cells (TAMs) corresponding to vascular changes during GBM progression. As tumor vessels transitioned from the initial dense regular network to later scant and engorged vasculature, TAMs shifted away from perivascular regions and trafficked to vascular-poor areas. This process was heavily influenced by the immunocompetence state of the host. Utilizing a sensitive fluorescent UnaG reporter to track tumor hypoxia, coupled with single-cell transcriptomics, we revealed that hypoxic niches attracted and sequestered TAMs and cytotoxic T lymphocytes (CTLs), where they were reprogrammed toward an immunosuppressive state. Mechanistically, we identified chemokine CCL8 and cytokine IL-1β as two hypoxic-niche factors critical for TAM trafficking and co-evolution of hypoxic zones into pseudopalisading patterns. Therefore, perturbation of TAM patterning in hypoxic zones may improve tumor control.
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•Host immune status influences tumor vasculature and hypoxic-zone formation in GBM•Spatial patterning of TAM and CTL parallels hypoxic-zone maturation to pseudopalisades•Sequestered TAM and CTL in hypoxic zones are reprogrammed toward immunosuppression•TAM/CTL organization involves CCL8 and IL-1β as niche factors in hypoxic zones
Glioblastoma is notorious for immunosuppression, but the mechanisms are unclear. Sattiraju et al. report that hypoxic zones in GBM attract and sequester tumor-associated myeloid cells and cytotoxic T cells, where they are reprogrammed into an immunosuppressive state. This process is influenced by the immunocompetence state of the host and involves CCL8 and IL-1B as niche factors in hypoxic zones.
Abstract
Hypoxia (low oxygen) has been associated with adverse effects in tumor biology by exaggerating the capabilities of invasion, proliferation, and survival of tumor cells within the tumor ...microenvironment. We engineered glioblastoma (GBM) proneural cells with a novel hypoxia reporter, HRE-UnaG, to study areas of tumor hypoxia and the effects that these hypoxic cells have on tumorigenesis. Single cell RNA-seq analysis from a mouse intracranially injected with our HRE dUnaG GBM cells revealed a shift to a mesenchymal state upon hypoxia (detected by expression of UnaG). Two genes, CXCR4 and NXPH4, were identified as being specifically induced in the hypoxic population. Our studies focus on the hypothesis that these two hypoxia induced genes, CXCR4 and NXPH4, are upregulated in hypoxic GBM cells, which may allow tumor cells to become more aggressive and resistant to conventional forms of therapies. GBM cells will be transduced with lentiviral vectors for Dox inducible shRNA knockdown of CXCR4 or NXPH4 to test specific contribution of these genes to the phenotype of the hypoxic population, with particular focus on the change in invasion and overall tumor burden upon gene silencing.
Abstract
Glioblastoma (GBM) is the most common malignant primary brain tumor and its invasive nature makes complete dissection difficult. However, how GBM invade in the brain is poorly understood. ...Although, microglia are the resident immune cells in the brain that can support GBM invasion, their roles are yet elusive. Here, we retraced early steps of GBM invasion and their interaction with tumor-associated microglia (TAM) in a highly infiltrative murine GBM model in immunocompetent background. We show that microglia are activated in a wide tumor field ahead of GBM invasion, followed by glial net formation, encircling invading tumors. Next, physical contacts with GBM cells initiate an astounding morphological, and functional transformation of microglia and marrow-derived macrophages to form collectively organized migration streams with intertwined GBM cells. Here we show that Kdm6b, a H3K27me3 demethylase, is induced in TAMs during this process. Intriguingly, GBM disseminated more widely in juvenile host brains, which was associated with highly induced Kdm6b expression. Moreover, we show that Kdm6b inhibition with small chemical inhibitor GSKJ-4, perturbs TAM reprogramming, and GBM invasion. Together, our data reveal that microglia reprogramming provide directional cues for GBM invasion through epigenetic mechanisms and this may give insights to halt GBM invasion.
Abstract
Glioblastoma (GBM) is the most common and lethal brain cancer that invariably recurs after therapy due to presence of resistant GBM cells within hypoxic and peri-necrotic regions. ...Eradicating such GBM cells, which constitute a major source of tumor recurrence, is important to curb disease relapse. An endogenously expressed, spatially sensitive hypoxia reporter would therefore be a valuable tool to evaluate hypoxic zones in GBM in detail, and to measure the efficacy of hypoxia-activated drugs. For this purpose, we engineered a lentiviral vector that carries a hypoxia reporter, consisting of HIF response elements (HRE) that drive expression of UnaG fluorescent protein, which fluoresces independent of oxidative maturation. We validated the sensitivity of our reporter in vitro using U87MG, GBM2, and patient-derived GBM stem cell lines, and we performed intracranial transplantations of GBM cells in SCID mice to identify cells undergoing hypoxic stress in in vivo microenvironment. In addition, GL261 murine GBM cells with hypoxia reporter were intracranially implanted in C57BL/6 mice as syngeneic model for studies on immune responses. Brains from our transplant studies were dissociated and single-cell RNA sequencing (Drop-Seq) was performed to investigate heterogeneity in response to hypoxia within GBM cells and the cellular composition of microenvironment. We will also apply a hypoxia-activated prodrug, Evofosfamide (Evo), in our ongoing studies that can potentially eradicate hypoxic tumor cells and increase T cell infiltration and reverse immune suppression. As hypoxic niches are thought to confer resistance to radiation therapy (XRT), combining XRT with Evo could thus improve therapy efficacy. Our hypoxia gene reporter, combined with single-cell transcriptomics, could therefore serve as an effective tool to enable fundamental investigation of GBM microenvironment and could be used to evaluate therapies targeting tumor microenvironment to enhance GBM patient survival.
Abstract
Glioblastoma (GBM) is the most common malignant primary brain tumor. The nature of invasiveness of GBM makes complete surgical resection difficult. However, how GBM cells achieve wide ...infiltration in the brain is poorly understood. Microglia, the resident immune cells in the brain can support GBM growth and invasion, but the underlying mechanisms remain elusive. Here, we show that microglia are activated in a wide field away from tumor boundaries, ahead of tumor cell infiltration. Invading GBM cells are in close contact with microglia, progressively aligned with one another in the direction of tumor invasion. Moreover, ECM is also aligned with the infiltrating tumor and microglia, which may serve as invasion tracks in the brain. Mechanistically, we demonstrate that microglia direct cellular alignment and ECM remodeling in the invasion tracks through an axon guidance receptor Plexin-B2. Myeloid-specific ablation of Plexin-B2 perturbs microglia and tumor cell alignment, microglia migration, ECM organization, and GBM invasiveness. Together, our data reveal a hitherto under-appreciated role of microglia in providing directional cues for GBM invasion through physical interaction and alignment of ECM and tumor cells, thus providing new insights and novel molecular targets in curbing GBM invasion.
Glioblastoma (GBM) is the most common primary malignant astrocytoma associated with a poor patient survival. Apart from arising de novo, GBMs also occur due to progression of slower growing grade III ...astrocytomas. GBM is characterized by extensive hypoxia, angiogenesis, proliferation and invasion. Standard treatment options such as surgical resection, radiation therapy and chemotherapy have increased median patient survival to 14.6 months in adults but recurrent disease arising from treatment resistant cancer cells often results in patient mortality. These treatment resistant cancer cells have been found to exhibit stem cell like properties. Strategies to identify or target these Glioblastoma Stem Cells (GSC) have proven to be unsuccessful so far. Studies on cancer stem cells (CSC) within GBM and other cancers have highlighted the importance of paracrine signaling networks within their microenvironment on the growth and maintenance of CSCs. The study of GSCs and their communication with various cell populations within their microenvironment is therefore not only important to understand the biology of GBMs but also to predict response to therapies and to identify novel targets which could stymy support to treatment resistant cancer cells and prevent disease recurrence. The purpose of this chapter is to introduce the concept of GSCs and to detail the latest findings indicating the role of various cellular subtypes within their microenvironment on their survival, proliferation and differentiation.
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